Project Summary Channelopathies are prominent in a variety of neurological and psychiatric disorders. Variants of SCN2A (encoding neuronal channel NaV1.2) have been associated with infantile epilepsy, epileptic encephalopathy, autism spectrum disorder, and developmental disorders with heterogeneous clinical manifestations. The functional consequences of most SCN2A variants identified by clinical sequencing are not known, and their contribution to the manifested clinical phenotypes remains unclear. Patch clamp recording has been the cornerstone of research determining the pathogenicity of ion channel variants; however, the technique is time-, labor- and resource-intensive. The lack of functional data for the majority of clinically identified SCN2A and failure to explicitly incorporate information about the ascertainment and prior probability can lead to misdiagnoses (if a benign variant is presumed pathogenic) and overestimation of penetrance if modestly functional variants are systematically excluded from the genetic interpretation. It is therefore imperative to design a scalable strategy to comprehensively classify functional categories of all possible variants for SCN2A to help dissect the genotype and clinical phenotype relationship and inform clinical practice. This proposal takes advantage of MITE (Mutagenesis by Integrated TilEs) technology to generate a library that contains every possible synonymous and nonsynonymous variant in hotspot regions of SCN2A coupled with a robust cellular readout and a pooled screen to classify all variants into functional categories. To our knowledge, this is the first study to systematically annotate the function of a comprehensive set of all possible missense and synonymous variations in a channelopathy. The goal of this pilot study is to demonstrate proof-of-principle that we can create a comprehensive substitution lookup map for either LOF or GOF variants in SCN2A, and validate an allele-characterization framework that can be scaled to meet the goals of genome-guided genotype-phenotype mapping in the rest of SCN2A.